Vol 13 No 01 January-February 2016 2471Vol 13 No 01 January-February 2016 2527 UROLOGICAL ONCOLOGY Evaluation of Vitronectin Expression in Prostate Cancer and the Clinical Significance of the Association of Vitronectin Expression with Prostate Specific Antigen in Detecting Prostate Cancer Yue Niu,1 Ling Zhang,2 Xing Bi,1 Shuai Yuan,1 Peng Chen1* Purpose: To detect the expression of vitronectin (VTN) in the tissues and blood serum of prostate cancer (PCa) patients, and evaluate its clinical significance and to evaluate the significance of the combined assay of VTN and prostate specific antigens (PSA) in PCa diagnosis. Materials and Methods: To detect the expression of VTN as a potential marker for PCa diagnosis and prognosis, immunohistochemistry was performed on the tissues of 32 patients with metastatic PCa (PCaM), 34 patients with PCa without metastasis (PCa), and 41 patients with benign prostatic hyperplasia (BPH). The sera were then sub- jected to Western blot analysis. All cases were subsequently examined to determine the concentrations of PSA and VTN in the sera. The collected data were collated and analyzed. Results: The positive expression rates of VTN in the tissues of the BPH and PCa groups (including PCa and PCaM groups) were 75.61% and 45.45%, respectively (P = .005). VTN was more highly expressed in the sera of the BPH patients (0.83 ± 0.07) than in the sera of the PCa patients (0.65 ± 0.06) (P < .05). It was also more highly expressed in the sera of the PCa patients than in the sera of the PCaM patients (0.35 ± 0.08) (P < .05). In the diagnosis of BPH and PCa, the Youden indexes of PSA detection, VTN detection, and combined detection were 0.2620, 0.3468, and 0.5635; the kappa values were 0.338, 0.304, and 0.448, respectively, and the areas under the receiver operating characteristic curve were 0.625, 0.673, and 0.703 (P < .05), respectively. Conclusion: VTN levels in sera may be used as a potential marker of PCa for the diagnosis and assessment of disease progression and metastasis. The combined detection of VTN and PSA in sera can be clinically applied in PCa diagnosis. Keywords: cell line; tumor; prostatic neoplasms; vitronectin; metabolism; humans; biomarkers; early detection of cancer; prostate-specific antigen. INTRODUCTION Prostate cancer (PCa) is a malignant tumor common among male Europeans and Americans. The inci- dence of this disease in China has gradually increased in recent years.(1) Prostate specific antigen (PSA) is the most widely used prostate tumor markers. Serum PSA concentrations significantly affect treatment modalities in males with PCa. However, PSA tend to increase dur- ing benign prostate hyperplasia (BPH), prostatitis, ure- thral catheter, and digital rectal exams.(2) Over-diagno- sis and the resulting over-treatment of occult cancer are thus common. PSA detection suffers from limited sen- sitivity and specificity. Furthermore, the significance of PSA declines in the later stages of the disease.(3,4) The search for effective serum tumor markers for PCa is sig- nificant in improving the level of the diagnosis of PCa and determining treatment prognosis. Vitronectin (VTN) is a glycoprotein, which is a member of the integrin family. It changes in the sera and tissues of people, and is associated with the occurrence and metastasis of tumor.(5) However, the studies on VTN and PCa are relatively rare. Hence, the present study aimed to explore the expression of VTN in the tissues and sera of PCa patients and the significance of the combined detection of VTN and PSA in the diagnosis and treatment of PCa. MATERIALS AND METHODS Study Patients Patient data, blood samples, and prostate tissue samples were collected from the prostate disease patients of the urology department of the Xinjiang Medical Universi- ty Affiliated Tumor Hospital between December 2010 and February 2015. All cases were examined for PSA. 1 Affiliated Tumor Hospital of Xinjiang Medical University, Xinjiang, China. 2 Center for Disease Control and Prevention of Xinjiang Uygur Autonomous Region, Xinjiang, China. *Correspondence: Affiliated Tumor Hospital of Xinjiang Medical University, Xinjiang, China. Tel: +88 991 7819152. Fax: +88 991 7968111. E-mail: alex-new@163.com. Received June 2015 & Accepted November 2015 10% ≤ color area < 50%, 2 points; 50% ≤ color area < 75%, 3 points; color area ≥ 75%. Color intensity was multiplied with color area; a score of 3 or more was considered as positive, whereas a score of less than 3 was considered as negative. Western Blot Analysis of VTN in Sera Western blot analysis was carried out according to the instruction manual, which provided guidelines regard- ing the preparation of sodium dodecyl sulfate (SDS)-po- lyacrylamide gel electrophoresis (PAGE), addition of samples, electrophoresis, transfer, sealing, addition of the first antibody (Mouse Anti-Human, R&D Co., 614 McKinley Place NE Minneapolis, MN 55413, USA), cleaning, addition of the second antibody (Rabbit An- ti-Mouse, Sigma Co., St. Louis, MO, USA), cleaning, chemiluminescence, exposure, development, and so on. Pictures of the chromogenic results were taken with an image analysis system, which was used to record the optical density and intensity values of the electrophoret- ic bands. The calculation formula for the optical density ratios was (intensity value of VTN band × optical densi- ty value of VTN band) / (intensity value of β-actin band × optical density value of β-actin band). The analysis was semi-quantitative. Expression of Vitronectin in Patients with PCa-Niu et al. Urological Oncology 2528 Pathological types were determined via biopsy or oper- ation, whereas metastasis was determined via comput- ed tomography (CT) scan, magnetic resonance imaging (MRI) and bone scan. Finally, 34 patients with pros- tate cancer without metastasis (PCa), 32 patients with prostate cancer with metastasis (PCaM), and 41 patients with BPH were included. The mean ages of the patients with PCa, PCaM, and BPH were 65.27 ± 9.07, 67.02 ± 10.02, and 64.12 ± 7.33 years, respectively. Immunohistochemistry for VTN in Tissues All tissue samples were collected via surgery or bi- opsy. Multiple 4-µm-thick sections of representative formalin-fixed, paraffin-embedded tissues were cut for immunohistochemical studies. A polymer-based immunohistochemical was used to detect VTN (Mouse Anti-Human, R&D Co, 614 McKinley Place NE Min- neapolis, MN 55413, USA). All immunostained sec- tions were examined under a light microscope (Olym- pus CKX41, Olympus Optical Co., Ltd., Tokyo, Japan) to evaluate VTN. VTN staining was cytoplasmic. The standards for determining immunohistochemis- try were as follows. Color intensity: 0 point; no stain, 1 point; faint cytoplasmic stain, 2 points; diffuse cy- toplasmic stain, 3 points; diffuse intense cytoplasmic stain. Color area: 0 point; color area < 10%, 1 point; Table 1. Vitronectin expression in prostate cancer tissues and its relationship with clinicopathological factors. Factor Positive, no. Negative, no. Expression Rate, % χ2 P Value Age, years < 70 16 19 45.16 0.002 .964 ≥ 70 14 17 45.71 PSA, ng/mL < 4 2 0 100.00 11.24 .004 4-10 11 3 78.57 > 10 17 33 34.00 Clinical stage T2 7 5 58.33 7.41 .025 T3 13 7 65.00 T4 10 24 29.41 Gleason score ≤ 6 8 2 80.00 11.06 .004 7 12 8 60.00 ≥ 8 10 26 27.78 Tumor metastasis Yes 9 23 28.12 7.52 .006 No 21 13 61.76 Abbreviation: PSA, prostate specific antigen. Vol 13 No 01 January-February 2016 2529 Determination of the Concentrations of VTN and PSA in Sera The VTN concentrations in the sera of all the patients were detected via an enzyme-linked immunoassay (Synergy 2 multimode microplate reader, BioTek Co., USA; Enzyme-linked immunoassay kit, R&D Co., 614 McKinley Place NE Minneapolis, MN 55413, USA), whereas the PSA concentrations were detected via an electrochemiluminescence immunoassay (Cobas 6000 fully automatic electrochemistry luminescence instru- ment and corresponding kit, Roche Co., Grenzach, Ger- many). The positive results of the combined parallel detection indicated that PSA or VTN was positive in the PCa patients. Statistical Analysis Data analysis and processing were performed with the Statistical Package for the Social Science (SPSS Inc, Chicago, Illinois, USA) version 16.0. The immunohis- tochemistry results, the VTN expression in PCa tissue, and the relationship of such expression with clinico- pathological factors were analyzed with the chi-square test. The Western blot results were obtained via vari- ance analysis and the Student-Newman-Keuls (SNK) method, α = 0.05. The evaluation indexes of VTN detection, PSA detection, and combined parallel detec- tion in the sera from the PCa and BPH groups included kappa values, sensitivity, specificity, Youden indexes, and total coincidence rates. A receiver operating char- acteristic (ROC) curve was drawn using the sensitivity and specificity indexes to compare VTN detection, PSA detection, and their combined parallel detection in sera for PCa diagnosis. RESULTS Immunohistochemistry of VTN in Tissue Sam- ples and its Relationship with Clinicopathological Factors VTN protein was expressed in PCa tissues (including PCa and PCaM tissues) and BPH tissues, as shown in Figures 1 and 2, respectively. The cytoplasm was stained brown, as shown in both figures. According to the immunohistochemical results, the positive ex- pression rates of VTN in the BPH and PCa groups (in- cluding the PCa and PCaM groups) were 75.61% and Table 2. Comparison of experimental diagnosis indexes of sera with and without metastatic prostate cancer. Test Index Sensitivity % Specificity % Total coincidence rate, % Youden index VTN detection 56.90 77.78 64.89 0.3468 PSA detection 76.75 49.45 59.57 0.2620 Combined parallel detection 80.23 76.12 67.12 0.5635 Abbreviations: VTN, Vitronectin; PSA, prostate specific antigen. Figure 1. Vitronectin expression in tissue samples of patients with benign prostatic hyperplasia. A) No stain (0 point); B) Faint cytoplasmic stain (1 point); C) Diffuse cytoplasmic stain (2 points); D) Diffuse intense cyto- plasmic stain (3 points). Figure 2. Vitronectin expression in tissue samples of patients with and without metastatic prostate cancer. A) No stain (0 point); B) Faint cyto- plasmic stain (1 point); C) Diffuse cytoplasmic stain (2 points); D) Diffuse intense cytoplasmic stain (3 points). Expression of Vitronectin in Patients with PCa-Niu et al. 45.45%, respectively. The VTN protein expression in the BPH group was higher than that in the PCa group (including PCa and PCaM; P = .005). The VTN expression in the PCa tissues (including PCa and PCaM tissues) was related to the PSA of newly diagnosed patients, clinical stage, Gleason score, and tumor metastasis; however, it was not associated with patient age (Table 1). Western Blot Analysis of VTN in Serum Samples Figure 3 shows that VTN could be detected in the se- rum samples from the PCaM, PCa, and BPH groups. Specific bands appeared with a relative molecular mass of 72 kDa. The optical density ratios of VTN in the serum samples from the BPH, PCa, and PCaM groups were 0.83 ± 0.07, 0.65 ± 0.06, and 0.35 ± 0.08, respec- tively. The results of the variance analysis showed that the optical density ratios of VTN in the serum samples from the BPH, PCa, and PCaM groups were not to- tally equal (F = 396.72, P = .000). The results of the SNK method showed that VTN expressions in the three groups differed. The optical density ratios of VTN in the serum samples from the BPH group were higher than those from the PCa group. In addition, VTN ex- pression in the PCa group was higher than that in the PCaM group (Figure 4). Combined Parallel Detection of VTN and PSA for PCa and BPH Diagnosis The VTN concentrations in the BPH, PCa, and PCaM groups were 219.63 ± 25.30, 201.72 ± 19.37, and 170.05 ± 23.80 ng/mL, respectively, with statistically signif- icant differences (P < .05). The PSA concentrations in the BPH, PCa, and PCaM groups were 5.45 ± 3.48, 15.45 ± 9.66, and 65.29 ± 31.50 ng/mL, respectively, with statistically significant differences (P < .05). Kappa values were used to evaluate the reliability of the diagnostic tests. The kappa values of VTN detection, PSA detection, and combined parallel detection in the PCa and PCaM groups were 0.338, 0.304, and 0.408, respectively. According to Kanidis and Koch’s stand- ards, combined parallel detection was found to be more consistent than single detection in the PCa and PCaM groups, with such consistency being moderate. It yield- ed a certain value for PCa diagnosis. Sensitivity, specificity, the Youden index, and the total coincidence rate were analyzed. The calculated results showed that the single detection of PSA in PCa exhib- ited high sensitivity but low specificity and total coin- cidence rate. By contrast, the single detection of VTN yielded opposite results. In addition, the specificity of the combined parallel detection of PSA and VTN was Figure 3. Vitronectin expression detected by Western blot analysis. Figure 3 shows that vitronectin could be detected in the serum samples from the PCaM, PCa, and BPH groups. Specific bands of vitronectin appeared with a relative molecular mass of 72 kDa, and β-actin appeared with 42 kDa. Abbreviations: BPH, benign prostatic hyperplasia; PCa, prostate cancer; M, metastasis. 1: BPH; 2: PCa; 3: PCaM. Figure 4. Optical density ratios of vitronectin for serum samples from each group. Figure 4 shows that the optical density ratios of vitronectin in the serum samples from the BPH, PCa, and PCaM groups were 0.83 ± 0.07, 0.65 ± 0.06, and 0.35 ± 0.08, respectively. The results were statistically signifi- cant. Abbreviations: BPH, benign prostatic hyperplasia; PCa, prostate cancer; M, metastasis. Expression of Vitronectin in Patients with PCa-Niu et al. Urological Oncology 2530 Vol 13 No 01 January-February 2016 2471Vol 13 No 01 January-February 2016 2531 slightly lower than that of single PSA detection. How- ever, the sensitivity, total coincidence rate, and Youden index were better in the combined approach (Table 2). The values of PSA and VTN detection for PCa diag- nosis were evaluated with the ROC curve. The areas under the curves (AUCs) of PSA and VTN were 0.625 and 0.673 in the PCa and PCaM groups (Figure 5), re- spectively; the difference was statistically significant (P < .05). The AUC of the combined parallel detection of VTN and PSA was 0.703 in both the PCa and PCaM groups (Figure 5); thus, the accuracy of this combined detection method is better than that of single detection (P < .05). DISCUSSION The VTN protein is a specific ligand of the αvβ3 in- tegrin family that is present on the surface of vascular endothelial cells.(6) because it contains the RGD pep- tide sequence (Arg-Gly-Asp). The interaction of VTN and αvβ3 promotes the adhesion of monocytes and endothelial cells in blood circulation. αvβ3 is widely expressed on the surface of malignant tumor cells and vascular endothelial cells in different tissues; this mark- er promotes malignant biological behavior, such as the occurrence, development, angiogenesis, invasion, and metastasis of malignant tumors.(7–9) The high expression of matrix metalloproteinases (MMPs) is closely relat- ed to the invasion and metastasis of malignant tumors. (10) αvβ3 can interact with MMPs and decompose fi- bronectin, as well as promote the invasion and metasta- sis of various malignant tumor cells.(11) In serum protein chips of hepatocellular carcinoma patients, VTN was degraded into small peptides with MMP-2; the outcome confirmed that small peptides of VTN could be used for the serological diagnosis of liver cancer.(12) In addi- tion, VTN interacts with urokinase receptors (uPARs), which control cell adhesion and migration.(13) To date, studies on the relationship between VTN and PCa are relatively rare. The present study is the first to detect VTN protein expression in PCa and BPH tissues via immunohistochemistry. According to the immunohistochemical results, the positive expression rates of VTN in the BPH and PCa groups (including PCa and PCaM groups) were 75.61% and 45.45%, respectively. The expression of VTN protein was the highest in the BPH group, followed by the PCa group. Simultaneously, the expression of VTN in patient sera was detected via Western blot analysis, the results of which are consistent with the immunohistochemistry results. This outcome showed that VTN in the serum samples from the BPH group was higher than that in the serum samples from the PCa group. Moreover, the VTN expression in the PCa group was higher than that in the PCaM group. In addition, the VTN expression in the PCa tissues (including PCa and PCaM tissues) was related to the PSA of newly diagnosed patients, clinical stage, Gleason score, and tumor metastasis. Therefore, VTN may participate in the occurrence and development of PCa via cell adhesion and migration. As a member of the integrin family, VTN promotes the adhesion of monocytes and endothelial cells in blood circulation. VTN also interacts with uPAR to adjust cell adhesion and migration, which are directly involved in PCa metastasis. VTN can interact with MMPs to pro- mote the invasion and metastasis of PCa. With an in- creased expression of αvβ3, uPAR, and MMPs during the occurrence and mineralization process of PCa, VTN combines with αvβ3, interacts with uPAR, and is de- graded by MMPs, thereby gradually reducing the VTN content in serum. Therefore, VTN levels in sera may be used as a potential marker of PCa for the diagnosis and assessment of disease progression and metastasis. In addition, the analysis of PSA and VTN concentra- tions in sera and their ROC curves for the PCaM and PCa patients showed that the combined parallel detec- tion of VTN and PSA achieved better diagnostic accu- racy than the PCa detection method based on a single index. Therefore, combined parallel detection can be Figure 5. Receiver operating characteristic curves of prostate specific an- tigen detection, vitronectin detection, and combined parallel detection of vitronectin and prostate specific antigen. Figure 5 shows that the areas under the receiver operating characteristic curves of prostate specific antigen detection, vitronectin detection, and combined parallel detection of vitronectin and prostate specific antigen were 0.625, 0.673 and 0.703 for the prostate cancer and metastatic pros- tate cancer groups; the difference was statistically significant (P < .05). Hence, the accuracy of combined parallel detection is better than that of single detection. Expression of Vitronectin in Patients with PCa-Niu et al. clinically applied in PCa diagnosis. The results of this study provide only preliminary clues. The number of samples was limited, and no follow-up was conducted after radical operation of PCa. There- fore, larger samples and long-term follow-up are need- ed to confirm the role of VTN detection in serum to diagnose and judge the prognosis of PCa. CONCLUSIONS The VTN expression in the sera and prostate tissues of the BPH patients was higher than that in the sera and prostate tissues of the PCa patients. VTN levels in sera may be used as a potential marker of PCa for the diag- nosis and assessment of disease progression and metas- tasis. The combined parallel detection of VTN and PSA in sera can be clinically applied in PCa diagnosis. CONFLICT OF INTEREST None declared. REFERENCES 1. Na YQ, Ye ZQ, Sun G. Chinese urology disease diagnosis and treatment guidelines. Beijing: People's Medical Publishing House; 2011. p. 49. 2. Xu Y, Zhang ZH. Prostate Cancer. Beijing: Science and Technology Literature Press; 2009. p. 115. 3. Partin AW, Carter HB, Chan DW, et al. Prostate specific antigen in the staging of localized prostate cancer: influence of tumor differentiation, tumor volume and benign hyperplasia. J Urol. 1990;143:747-52. 4. Özdemir E, Çiçek T, Kaya MO. Association of serum YKL-40 level with tumor burden and metastatic stage of prostate cancer. Urol J. 2012;9:568-73. 5. Paradis V, Degos F, Dargère D, et al. Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases. Hepatology. 2005;41:40-7. 6. Zhang CL, Wang RF, Zhang L, et al. (131)I labeling and bioactivity evaluation of a novel RGD diner targeted to integrin αvβ3 receptor. Beijing Da Xue Xue Bao. 2011;43:295-300. 7. Ai WB, Liu XY, Xiong ZY, et al. Effect of integrin αvβ3 on cell proliferation and invasive ability of C6 glioma in vitro. Chin J Cancer Prev Treat. 2007;14:1450-3. 8. Reuning U. Integrin αvβ3 promotes vitronectin gene expression in human ovarian cancer cells by implicating rel transcription factors. J Cell Biochem. 2011;112:1909-19. 9. Pola C, Formenti SC, Schneider RJ. Vitronectin-αvβ3 integrin engagement directs hypoxia-resistant mTOR activity and sustained protein synthesis linked to invasion by breast cancer cells. Cancer Res. 2013;73:4571-8. 10. Ni XG, Bai XF, Wang GQ, et al. Clinical significance of expressions of MMP-2 and PCNA in pancreatic cancer tissues. Chin J Cancer Prev Treat. 2011;18:108-11. 11. Jiao Y, Feng X, Zhan Y, et al. Matrix metalloproteinase-2 promotes αvβ3 integrin- mediated adhesion and migration of human melanoma cells by cleaving fibronectin. PLoS One. 2012;7:e41591. 12. Paradis V, Degos F, Dargère D, et al. Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases. Hepatology. 2005;41:40-7. 13. Rea VE, Lavecchia A, Di Giovanni C, et al. Discovery of new small molecules targeting the vitronectin-binding site of the urokinase receptor that block cancer cell invasion. Mol Cancer Ther. 2013;12:1402-16. Expression of Vitronectin in Patients with PCa-Niu et al. Urological Oncology 2532